The zona pellucida (ZP) is a complex extracellular glycoprotein matrix that surrounds the mature mammalian oocyte and preimplantation embryo. The ZP3 protein has special functional significance as it confers upon the oocyte the ability to recognize and bind sperm. Only very limited and inconclusive data exists regarding the origin and development of the ZP and the hormonal regulation of ZP3 gene transcription. Using the techniques of competitive RT-PCR and immunocytochemistry, this laboratory has demonstrated for the first time that cultured human luteinized granulosa cells, in addition to the oocyte, produce ZP3 mRNA and protein, and moreover that ZP3 gene transcription is stimulated by FSH. This raises the intriguing possibility that granulosa cells may contribute to the formation of the ZP, or may serve other granulosa cell specific functions within the developing follicle. It is the goal of this study to develop a radioimmunoassay for the quantification of ZP3 protein, and to use this assay with RT-PCR and immunotycochemistry to delineate the localization and regulation of ZP3 mRNA and protein in the human granulosa cell. The effect of purified recombinant ZP3 protein upon granulosa cell steroidogenesis will also be examined. Our long term objective is to discover potential new roles for this protein in folliculogenesis.